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Application of gene OsERL in reducing stomatal density of plant

A gene and aspect technology, applied in the directions of botanical equipment and methods, applications, plant products, etc., to achieve the effect of reducing stomatal density, improving water use efficiency and drought stress tolerance

Inactive Publication Date: 2012-07-04
山东省农业科学院高新技术研究中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the application of this kind of gene in drought resistance genetic engineering has not been reported yet.

Method used

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  • Application of gene OsERL in reducing stomatal density of plant
  • Application of gene OsERL in reducing stomatal density of plant
  • Application of gene OsERL in reducing stomatal density of plant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Embodiment 1: Isolation and cloning are used to construct the DNA fragment of OsERL gene plant expression vector

[0021]Total RNA was extracted from leaves of rice variety Nipponbare (a publicly reported variety) using TRIZOL reagent (Invitrogen). The specific steps are as follows: put 20 mg leaves into a liquid nitrogen pre-cooled mortar, add liquid nitrogen to quickly grind into powder, put the powder into a 1.5ml centrifuge tube, quickly add 1ml Trizol (Invitrogen) and invert to mix well, stand at room temperature Leave for 5 minutes. Centrifuge at 12,000 rpm for 10 minutes at 4°C, and transfer the supernatant to a new 1.5ml centrifuge tube. Add 200 μl of chloroform, shake vigorously by hand for 15 seconds, and let stand at room temperature for 2-3 minutes. Centrifuge at 12000 rpm for 15 minutes at 4°C. Take the colorless aqueous phase into a new 1.5ml centrifuge tube, add 250μl isopropanol, 250μl high salt solution, invert and mix well, and let stand at room tem...

Embodiment 2

[0023] Embodiment 2: Construction and genetic transformation of OsERL gene plant expression vector

[0024] In order to better analyze the function of OsERL, the applicant increased the expression level of OsERL gene in tobacco through overexpression technology. The function of the gene was studied according to the phenotype and physiological characteristics of the transgenic plants.

[0025] The construction method of the OsERL gene plant expression vector is as follows: first, the positive clone pMD18-OsERL cDNA obtained in Example 1 is double-digested with BamHI and SpeI, and the insert fragment is recovered; similarly, the plant expression of pCAMBIA1390-ubi is digested with the same method. vector, recovery of vector fragments. Use the recovered insert and vector fragments for ligation to transform Escherichia coli XL1-Blue. Positive clones were screened by enzyme digestion to obtain a plant expression vector named pCAMBIA1390-ubi-ERL (see figure 1 ). pCAMBIA1390-ubi ...

Embodiment 3

[0062] Example 3: Detection of transcript levels of OsERL gene in transgenic tobacco plants and wild-type tobacco

[0063] Tobacco wild-type SR varieties and 5 independent T3 generation transgenic tobacco plants were used as materials, RNA was extracted from tobacco leaves at the 5-leaf stage, and the transcript level of OsERL gene in tobacco leaves was detected by RT-PCR. The specific method is as follows: using TRIZOL reagent (Invitrogen) to collect 0.05g of tobacco leaves to extract total RNA from the above materials, and reverse-transcribe it into cDNA using reverse transcriptase SuperScript II (Invitrogen). The specific steps are as described in Example 1. Using the specific upstream primer OsERLRTF (5'-CGC AGA CAA CAC GGT CAT GG-3', SEQ ID NO: 5) of the OsERL gene, and the downstream primer OsERLRTR (5'-AAG AGC TCC GCA TCT GAC GC-3', SEQ ID NO: 6). At the same time, the specific upstream primer ACTINF (5'-TCC GGC GAC GGT GTC TCACA-3', SEQ ID NO: 7) of tobacco ACTIN gene a...

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Abstract

The invention discloses the application of a gene OsERL in reducing the stomatal density of a plant, belonging to the technical field of molecular biology. In the invention, through using a PCR (polymerase chain reaction) method, the full-length coding region of a gene OsERL of Nipponbare (a rice variety) is amplified, the gene OsERL is positively connected to a plant expression vector pCAMBIA1390-ubi; then the gene OsERL is subjected to genetic transformation so as to transform the gene OsERL into a tobacco plant, and the expression of the gene OsERL is improved, thereby obtaining a transgenic tobacco plant with enhanced gene OsERL expression. Experiments show that the over-expression of gene OsERLs in transgenic tobacco plants can promote the expansion of epidermal cells, thereby causing the decrease in stomatal density, the reduction of transpiration rate and water loss rate, and the enhancement of drought stress tolerance.

Description

technical field [0001] The invention relates to the application of the gene OsERL in reducing the stomatal density of plants, thereby leading to the enhancement of drought stress tolerance, and belongs to the technical field of molecular biology. Background technique [0002] Drought has become a worldwide problem. The world's arid and semi-arid areas have accounted for more than 1 / 3 of the land area. The impact of drought on plants ranks first among many natural adversity factors. Food loss caused by drought accounts for more than half of all natural disaster food losses. The Second National Agricultural Census Leading Group Office of the State Council, the Ministry of Land and Resources and the National Bureau of Statistics issued the Second National Agricultural Census Main Data Bulletin (No. 6) on February 29, 2008. More than half of the cultivated land is dry land. Under natural conditions, drought stress not only seriously affects the growth and yield of crops, but a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/54C12N15/82A01H5/00
Inventor 周晋军谢先芝刘婧钱凤芹王莹莹
Owner 山东省农业科学院高新技术研究中心
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